Methods for plant seed production

ABSTRACT

The invention provides methods for producing seeds in watermelon. In one embodiment methods are provided comprising grafting of a seed parent onto a stress tolerant rootstock, pollinating the seed parent with pollen from a pollen donor, and cultivating the seed parent until seed is formed. In specific embodiments, triploid seeds produced by a method of the invention are rendered conspicuously distinguishable from tetraploid seeds, and thus readily selected manually or by an automated machine. Methods for increasing seed yield and/or quality are also provided.

This application claims priority to U.S. Provisional Application No.61/093,131, filed on Aug. 29, 2008, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of seed production and, morespecifically, to methods of producing seeds of watermelon and otherspecies.

BACKGROUND OF THE INVENTION

Watermelons are natural diploids, referred to as 2N (N=11). Many plants,including watermelons, can undergo a duplication of their entire set ofchromosomes and exist as tetraploids 4N (4N=44). Watermelon tetraploidscan be produced routinely in the laboratory using cell biologytechniques.

A tetraploid parent can be crossed with a diploid parent to producetriploid seeds (3N=33). A hybrid triploid plant produces watermelonfruit which is “seedless,” meaning that it very rarely produces matureseeds, and only rarely produces immature seeds with hard seed coats, butno embryo. To obtain triploid watermelon seed, a cross between atetraploid and diploid line is made. The tetraploid female flower isused as the ‘seed parent’ and the diploid male flower is used as thepollen donor. Seeds obtained from this cross bear triploid embryos. Atetraploid seed parent typically produces only 5 to 10% as many seeds asa typical diploid plant. If female diploid flowers are pollinated withpollen coming from tetraploid flowers the result is empty seeds.

When production of triploid hybrids is done by open pollination, thereis no control on the source of pollen that reaches the stigma of thetetraploid flower, and thus both tetraploid and triploid seeds may befound in the same fruit. In the absence of selection methods, hybridseeds produced in this manner may therefore be contaminated with seedsresulting from self-pollination.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of producing hybridwatermelon seed comprising: (a) obtaining a first watermelon plant,wherein the plant has been grafted onto a rootstock that exhibitsresistance to at least one biotic and/or abiotic stress when understress conditions; (b) allowing the grafted first watermelon plant to bepollinated by a second watermelon plant to produce a hybrid watermelonseed; and (c) allowing hybrid watermelon seed to form. In oneembodiment, the first watermelon plant is a tetraploid watermelon plantthat produces triploid (3N) hybrid seeds after being pollinated by asecond watermelon plant that is diploid. In another embodiment, thefirst watermelon plant is a diploid watermelon plant that produces adiploid (2N) hybrid seed after being pollinated by a second diploidwatermelon plant. In some embodiments, the second diploid watermelon isgrafted onto a rootstock that exhibits a selected desirable phenotype.

A rootstock used in accordance with the invention may exhibit any typeof stress resistance. In one embodiment, the rootstock exhibitstolerance to an abiotic stress selected from the group consisting ofcold tolerance, heat tolerance, drought tolerance, and salt tolerance.In another embodiment, the rootstock exhibits biotic stress toleranceselected from the group consisting of disease resistance, insectresistance, and nematode resistance. Disease resistance includesresistance to bacterial, fungal, and viral diseases. In specificembodiments, the rootstock may be obtained from a plant selected fromthe group consisting of watermelon, squash, pumpkin, wax gourd, andbottle gourd. In further embodiments, the seed parent may or may not begrown under stress conditions.

Grafting carried out in accordance with the invention may be performedby any method known to one of skill in art. In one embodiment, graftingis performed by a method selected from the group consisting of splicegrafting, bud grafting, cleft grafting, side grafting, approachgrafting, and hole insertion grafting. Pollination may be carried out inaccordance with standard methods, including pollination by insects andhand-pollination.

In another aspect of the invention, methods are provided for producingtriploid seed, wherein the triploid seeds are rendered conspicuouslydistinguishable from the seeds of the tetraploid parent, such as basedon size and/or shape, particularly when seed parent is exposed to stressconditions during cultivation. Thereby, the invention allows selectingthe seeds of one ploidy category from the seeds of other ploidycategories based on size and/or shape. In one embodiment, the selectionmay be performed manually. In another embodiment, the selection may beperformed mechanically by an automated machine.

The invention further permits producing increased number of fruits,increased number of seeds per fruit, and increased total seed weight perfruit in a grafted seed parent relative to a non-grafted seed parent. Inspecific embodiments, the seed yield per fruit, including diploid andtetraploid plants, may be defined as, for example, at least about 5, 10,25, 50, at least about 75, at least about 100, at least about 125, atleast about 150, at least about 175, at least about 200, at least about225 or at least about 250 seeds per fruit, including from about 5 toabout 150, from about 34 to about 100, from about 60 to about 120, fromabout 50 to about 150, from about 100 to about 200, from about 125 toabout 250, and from about 150 to about 200, seeds per fruit produced byusing a grafted seed parent with a diploid pollen donor plant. In thecase of diploid watermelon plants, the seed yield per fruit may bedefined in specific embodiments as at least about 150, 200, 250, 300,350 or at least about 400.

In yet another aspect, the invention provides seeds and plants producedby a process that comprises crossing a first parent plant with a secondparent plant, wherein at least one of the first or second parent plantsis a plant grafted on a rootstock. In one embodiment of the invention,seed and plants produced by the process are first generation (F₁) hybridseeds and plants produced by crossing a plant in accordance with theinvention with another, distinct plant. Therefore, certain exemplaryembodiments of the invention provide an F₁ hybrid seed and plantthereof. In specific embodiments of the invention, a diploid watermelonvariety is used as the seed parent and the pollen donor is a seconddiploid watermelon plant. In other embodiments, the seed and pollenparents are a plant species selected from the group consisting of hotpepper, sweet pepper, tomato, squash, cucumber, pumpkin, gourd, melon,eggplant, and okra.

In still yet another aspect, the invention provides a population of seedparent and pollen donor parent plants in accordance with the invention.In one embodiment, a population of plants is provided planted inpollinating proximity in a field comprising a) tetraploid watermelonseed parent plants, wherein the plants have been grafted onto arootstock that exhibits stress tolerance when under stress conditions;and b) diploid watermelon pollen donor plants. In specific embodiments,the tetraploid watermelon seed parent plants and diploid watermelonpollen donor plants are planted in alternating rows, and may be plantedin a ratio of rows of tetraploid watermelon seed parent plants todiploid watermelon pollen donor plants of, for example, 1:1, 2:1, 3:1,4:1 or 5:1.

These and other features and advantages of this invention are describedin, or are apparent from, the following detailed description of variousexemplary embodiments of the devices and methods according to thisinvention.

DETAILED DESCRIPTION OF THE INVENTION

The invention overcomes limitations in the art by providing improvedmethods for producing watermelon seeds. In one embodiment of theinvention, the inventors surprisingly found that production of triploidseeds can be substantially improved by crossing a diploid pollen donorline with a tetraploid seed parent that has been grafted to a rootstockexhibiting resistance to biotic or abiotic stress when under stressconditions, and that these benefits are observed even in the absence ofexposure of the seed parent to stress to which the rootstock confersresistance. For example, use of a disease resistant rootstock was foundto confer improved seed production even in the absence of diseasepressure. Significantly, the inventors also found that triploid seedsproduced by the grafted seed parents exhibited a morphology that wasmore distinct from tetraploid seeds relative to ungrafted plants. Theimproved distinctions in seed morphological characteristics is of greatimportance due to the difficulty that can occur in distinguishing thetetraploid seeds also found in fruits of the seed parent from triploidseeds. By improving the distinctions between triploid and tetraploidseeds, such as in thickness and weight, the invention allows easyseparation of the triploid seeds by manual or automated screening. Seedquality and the efficiency of seed production is thereby increased,benefiting seed producers and farmers alike.

In accordance with the invention, grafted seed parents may also be usedto improve seed yield and quality. The inventors surprisingly found thatincreased numbers of seeds per fruit could be obtained in grafted seedparents relative to non-grafted seed parents, and even more surprisinglythe effect was found even in the absence of stress to which therootstock confers resistance. One embodiment of the invention thereforecomprises crossing a grafted seed parent, which may be a diploid ortetraploid watermelon plant, with a grafted or ungrafted pollen donorplant. Examples of diploid seed lines that may be used as seed parentsor pollen donors in accordance with the invention are well known in theart and include, but are not limited to, Allsweet, Crimson Sweet,Jubilee, Dixie Lee, Sugar Baby, Calsweet, Charleston Grey, and Minilee.

The techniques of the invention are also applicable to other specieswhich are amenable to grafting. Therefore the invention provides, in oneembodiment, a method of producing plant seed comprising: crossing afirst plant that has been grafted onto a rootstock that exhibits stresstolerance when under stress conditions, allowing the first plant to bepollinated by a second plant of the same species, and allowing seed toform on the first plant, wherein the first plant is grown in the absenceof said stress conditions. Examples of species applicable to the methodinclude hot pepper, sweet pepper, tomato, squash, cucumber, pumpkin,gourd, melon, eggplant, and okra. In one embodiment, the rootstockconfers resistance to bacterial, fungal and/or viral disease.

In another embodiment, a seed parent used in accordance with theinvention is a tetraploid plant that is crossed to a diploid pollendonor to produce triploid hybrid seed. Examples of tetraploid seedparents that may be used are known in the art. Methods for producingtetraploid watermelon plants are also known in the art and are describedin, for example, see Kihara, 1951 and Eigsti, 1971. To develop atetraploid, chemicals that alter mitosis of a diploid inbred line may beused so that unusual numbers of chromosomes are obtained. For example,colchicine is a chemical that alters the mitotic spindle fibers ofdiploid cells resulting in a number of cells that are tetraploid. Thediploid line used to create a tetraploid may be selected based on thetraits desired for the tetraploid line. Traits that are desired for atetraploid line may therefore first be introgressed into the diploidinbred lines that will be used to develop the tetraploid lines bybreeding methods well known to one of skill in the art. Thus, thediploid and tetraploid parent lines may be bred separately for thedesired traits.

Two generations of self-pollination and selection may be needed to “fix”the 4N condition because, after the colchicine treatment, chromosomalaberrations are often encountered that affect seed fertility, and mustbe eliminated. Once the stable tetraploid containing the desiredcharacteristics is verified, it then can be used as a stable femaleparent for the production of the triploid hybrid.

Crossing two different tetraploids followed by recombination breedingcan also result in new tetraploid lines. A longer breeding period isrequired to develop a stable tetraploid line using this approach. Thisis due to the larger number of genetic combinations and the fewer seedthat tetraploids produce.

Seed parents may also be propagated by tissue culture. The use of tissueculture to propagate watermelon plants is exemplified in Zhang et al.,1994a.

Selection and Use Rootstock

In another aspect, the invention provides methods involving selectingand using a rootstock having a desired phenotype comprising tolerance tostress when under stress conditions. In one embodiment it may bedesirable to select rootstock exhibiting desirable traits lacking in theseed parent. For instance, if a tetraploid seed parent is susceptible toa soil borne disease, a rootstock that is resistant to the soil bornedisease may be selected for grafting.

Examples of abiotic stress that rootstock may confer resistance toinclude, but are not limited to, cold, high temperature, salt, drought,and flood. Examples of biotic stress include, but are not limited to,plant pathogens, insects, and nematodes. Rootstocks may be from plantsof any ploidy level, including diploid and tetraploid plants. Examplesof known rootstocks include, but are not limited to, RS-841, Shintoza,Shogun, Charmtoza, FR Couple, and FR Strong.

It is not necessary that the rootstock be from the same species fromwhich the scion is derived. Rootstocks of other plants that arecompatible with the scion may also be used. Examples of such compatiblerootstocks for use with watermelon and other species include, but arenot limited to, bottle gourd (Lagenaria Siceraria var. hispida), waxgourd (Benincasa hispida), pumpkin (Cucurbita pepo), squash (Cucurbitamoschata), African horned cucumber (Cucumis metuliferus), Cucurbitamaxima, interspecific Cucurbita maxima×Cucurbita moschata andbur-cucumber Sicyos angulatus. (See e.g., Zhang et al., 1994a).

Rootstock may also be a progeny of an interspecific cross. In aparticular embodiment, the rootstock is a progeny of interspecific crossbetween Cucurbita maxima and Cucurbita moschata. An example of suchprogeny is Shintoza.

Grafting methods for watermelon and other species are known in the art.Examples of grafting methods include, but are not limited to, splicegrafting, side grafting, approach grafting, hole insertion grafting, budgrafting, and cleft grafting. (See e.g., Cushman 2006). The grafting maybe performed manually or by an automated machine.

Selecting Diploid Pollen Donors and Crossing with Seed parents

In particular embodiments of the invention, diploid pollen donor plantsare selected for use as a male parent in a cross with a graftedtetraploid or diploid plant. A stable diploid inbred may be selected.Adequate viable pollen supply from the diploid pollen donor may beneeded for the female flowers to set and develop into regular fruit. Insome embodiments, a diploid pollen donor is grafted onto a selectedrootstock. In other embodiments, the diploid pollen donor is anon-grafted plant. In one non-limiting embodiment, the diploid pollendonor plant may be a publicly available line such as, for example,Crimson Sweet, Jubilee, Sugar Baby, Dixie Lee, Allsweet, Calsweet,Charleston Grey, and Minilee. Generally pollen donors will be selectedthat produce progeny with desirable phenotypes when crossed with thecorresponding seed parent.

In accordance with the invention, processes are provided for crossing aseed parent with a pollen donor. These processes may, in specificembodiments, be further exemplified as processes for preparing hybridwatermelon seed or plants, wherein a seed parent is crossed with apollen donor watermelon plant of a different, distinct line to provide ahybrid that has a grafted watermelon plant as one of its parents. Inthese processes, crossing will result in the production of seed. Theseed production occurs regardless of whether the seed is collected.

In one embodiment of the invention, the first step in “crossing”comprises planting of seed parents and pollen donors in proximity sothat pollination will occur, for example, mediated by insect vectors. Insome embodiments, pollen may be transferred manually. The desired ratioof seed parents and pollen donors for watermelon hybrid seed productionis well known to one of skill in the art. In one example, the seedparents and pollen donors are planted at a ratio of 2:1. Examples ofother ratios of seed parents and pollen donors include, but are notlimited to, 1:1, 3:1, 4:1, and 5:1. In a particular embodiment, seedproduction fields may be planted at the ratio of 2 rows of female lineand 1 row of diploid male line.

A second step may comprise cultivating or growing the seeds of seedparent and pollen donor watermelon plants into plants that bear flowers.A third step may comprise preventing self-pollination of the plants,such as by emasculating the male portions of flowers, (i.e., treating ormanipulating the flowers to produce an emasculated parent watermelonplant). Self-incompatibility systems may also be used in some hybridcrops for the same purpose. Self-incompatible plants still shed viablepollen and can pollinate plants of other varieties but are incapable ofpollinating themselves or other plants of the same line. In someembodiments, all the male flower portions may be manually removed fromthe female plants. This process is known as de-budding or emasculation.In other embodiments, a male-sterile seed parent line may be used thatmay not require the de-budding process.

A fourth step for a hybrid cross may comprise cross-pollination betweenthe seed parent and pollen donor watermelon plants. In one embodiment,the pollen of the diploid pollen donor parent is transferred to a femalediploid or tetraploid seed parent flower by manual methods well known toone of in the art. In another embodiment, the pollen is transferred byinsect vectors. In a particular embodiment, the crossing of seed parentsmay involve self-pollination, which may occur manually or without anyhuman intervention.

Yet another step comprises harvesting seeds from the seed parent. All ora portion of the fruit set on the seed parent can be harvested, andseeds are isolated. (U.S. Pat. No. 6,018,101). The harvested seed can beused for any desired purpose, including grown to produce watermelonplants and sold to farmers.

Recovery of Seeds

Seeds may be recovered from matured fruits using methods known to one ofskill in the art. In some embodiments, an open pollinated tetraploidseed parent may produce tetraploid seeds in addition to triploid seeds.For example, in order to reduce the cost and labor involved in handpollination, the transport of the pollen may be left to bees in thefield of open pollinated seed parents. Under these conditions, sincethere is no control on the source of pollen that reaches the stigma ofthe tetraploid flower, tetraploid and triploid seeds may be found in thesame fruit.

In one example, a grafted tetraploid seed parent of the inventionproduces triploid seeds that are conspicuously distinguishable fromtetraploid seeds based on size and/or shape. In specific embodiments,the grafted seed parent yields seeds with improved distinctions in seedthickness and/or seed weight between triploid and tetraploid seedsrelative to a non-grafted seed parent. The grafted seed parent, even ifraised under a stress condition, yields triploid seeds that aredistinguishable from tetraploid seed and can be selected, for example,as being thinner than tetraploid seeds, weighing less than tetraploidseeds, and/or otherwise exhibiting morphological distinctions.

The triploid seeds may therefore be separated from the tetraploid seedsbased on differences in morphological characteristics. In oneembodiment, the seeds are manually separated. In another embodiment, theseeds are mechanically separated using an automated machine. Theautomated machine may separate the seeds in accordance with theirthickness and/or weight.

In a further embodiment, the invention provides a plant of a grafted andhybridized seed parent that exhibits an average seed number of at least50 seeds per fruit. In specific embodiments, the seed number may befurther defined as, for example, at least about 50, at least about 75,at least about 100, at least about 125 or at least about 150 seeds perfruit, including from about 50 to about 150, from about 34 to about 100,and from about 60 to about 120 seeds per fruit produced by using agrafted seed parent with a diploid pollen donor plant.

In another embodiment, the invention provides a plant of a grafted andhybridized seed parent that exhibits an average seed weight of at least8 grams (gms) per fruit. In specific embodiments, the seed weight may befurther defined as, for example, at least about 0.5 gms, at least about2 gms, at least about 5 gms, at least about 8 gms, at least about 10gms, at least about 20 gms or at least about 50 gms per fruit, includingfrom about 5 to about 50, from about 8 to about 40, and from about 8 toabout 15 gms per fruit produced by using a grafted seed parent with adiploid pollen donor plant.

Male Sterility

In certain aspects, a seed parent plant may be a male sterile (i.e.,comprise a male sterile scion). For example, a grafted tetraploidwatermelon scion as described herein maybe male sterile. In certainaspects, a seed parent may be rendered male sterile by physical removalof male plant tissues such as removal of pollen producing flowers. Insome aspects, a seed parent may be rendered male sterile by applicationof a gametocide, for example as described in U.S. Pat. No. 4,936,904. Instill further aspects, a seed parent may comprises a male sterilitytrait such as genetic or cytoplasmic male sterility. For example,tetraploid watermelon plants comprising a recessive gene for malesterility were described by Love et al., 1986. A stable male recessivewatermelon line, G17AB, has also been described (Zheng et al., 1994b;Zheng et al., 1996). Watermelon plants comprising a male sterility gene,such as the G17AB line, or progeny of such a plant or line of plants mayalso be used according to the methods described herein.

EXAMPLES Example 1 Effect of Grafting of Seed Parent on Seed Numbers andHybridity

Scions were obtained from the tetraploid watermelon hybrid variety TML110-1440 and grafted onto the rootstocks Charmtoza, Shintoza, andShogun. Grafting was carried out by side grafting. The graftedtetraploid was planted in a field together with non-grafted tetraploidplants and the diploid variety 110-6433 to serve as a male parent plant.The plants were grown under conditions in which the plants were notsubject to disease pressure or other stress of note.

Insect-mediated pollination was allowed to occur between the diploid andgrafted tetraploid plants and seeds were allowed to form. Seeds werethen harvested from the tetraploid parent and measurements taken of seedweight, seed number, and hybridity (% hybridity represent the % oftriploid seed in the fruit of the tetraploid parent). The results arepresented in Table 1

TABLE 1 Increased seed weight per fruit, seed number per fruit andhybridity resulted from grafting of seed Parents. Total Total Clean/Number Number Seed sized Total Grams of seeds of Weight Wt. Number perper Hybridity Fruits (Gms.) (Gms.) of seeds fruit fruit (%) Non Grafted35 348.53 347.80 5,015 9.96 143 65.40 Grafted (all) 60 689.90 691.209,753 11.50 163 81.83 Grafted 15 166.30 166.50 2,488 11.09 166 78.70Charmtoza Grafted 32 372.34 373.10 5,359 11.64 167 87.20 ShintozaGrafted 13 151.26 151.60 1,906 11.64 147 79.60 Shogun

As shown, in Table 1, the grafted plants yielded the total seed weightper fruit of 11.50 gms, but the non-grafted plants yielded the totalseed weight per fruit of only 9.96 gms. Also, the grafted plants yieldedan average of 163 seeds per fruit, but the non-grafted plants yielded anaverage of only 143 seeds. The grafted plants yielded the hybridity of81.83%, but the non-grafted plants yielded the hybridity of only 65.40%.

Example 2 Effect of Grafting of Seed Parent on Fruit Yield, Seed Weightand Hybridity

The tetraploid seed parent TML 110-1440 was grafted on the rootstocks,Shintoza, Shogun, Charmtoza, FR Couple, and FR Strong by side grafting.The grafted tetraploid plants, non-grafted tetraploid and the diploidpollen donor line 110-6433 were planted in a field and cultivated tomaturity. Diploid pollen donors were planted along with the graftedtetraploid seed parents, with spacings of 16″×16″, 32″×32″, 48″×48″, and24″×24″.

The tetraploid seed parents were open-pollinated by insects and seedswere allowed to form. Seeds were harvested and the seed weight, seednumber, and hybridity measured. The results are provided in Table 2.

TABLE 2 Increased seed weight per fruit, and hybridity resulted fromgrafting of seed parents. TSW Female parent, (Thousand Extended TML110-1440 Spacing # of Seed Weight) Yield plus Rootstock (inches)Gr./plant fruit/plant gms Hybridity Kg/Ac TML 110-1440 16 9.2 1.15 7087.2 60 w/o rootstock 32 15.1 1.69 71 85 49 48 18.5 1.57 70 89 40 24 131.35 70 83.7 56 Manually 4.2 85 self- pollinated Manually 7.6 72 cross-pollinated. Shintoza 16 14.5 1.45 83 94 32 30 2.5 67.95 88 98 48 43 3.8367.7 88 93 24 15.9 2.32 95.7 69 Manually N/A self- pollinated Manually6.4, 8.2 cross- pollinated Shogun 16 12.8 1.4 74 84 83 32 20.6 2.3 72 8667 48 N/A N/A 24 N/A N/A Manually 4.3, 7.8 76 & 80 self- pollinatedManually 20.9 72 cross- pollinated Charmtoza 16 16 1.7 72.05 83 104 3228.9 2.4 68 89 94 48 29.3 3.2 72.1 87 63 24 19.6 2.25 65.55 83.5 85Manually N/A self- pollinated Manually 8.4, 8.0 cross- pollinated FRCouple 16 17.6 1.75 70.7 114 (TW 1) 32 23.2 2.53 73.5 85 75 48 32.6 3.373.2 82 71 24 20.6 2.1 68.7 88.9 89 Manually 3.5 self- pollinatedManually 10.7, 8.8  cross- pollinated FR Strong 16 15.7 1.6 73.1 83.9102 (TW 2) 32 25 2.3 74.15 80 81 48 41.4 3.29 72.6 86 90 24 21.5 2.3867.75 83.3 93 Manually N/A self- pollinated Manually 12.8, 9.1  cross-pollinated

As shown in Table 2, the seed parents grafted on the rootstocksShintoza, Shogun, Charmtoza, FR Couple, and FR Strong yielded increasednumber of fruits, seed yield, total seed weight, and hybridity.

Example 3 The Effect of Grafting on Seed Numbers and Seed Yield

Tetraploid watermelon lines TML110-1440, TML110-0064, TCS110-1009, andTCS110-1018 were crossed as seed parent lines with a diploid pollendonor line. Prior to crossing tetraploid seed parents were grafted ontothe interspecific rootstock Cucurbita maxima×Cucurbita moschata(Shintoza) by side grafting. The diploid pollen donor plants wereungrafted.

Grafted and non-grafted tetraploid seed parent plants were transplantedon the same day into a field that was subject to normal (no stressnoted) conditions during cultivation. The diploid pollen donorWAS110-6433 was planted with the tetraploid seed parent plants. Theplants were grown to maturity with bee-mediated pollination of thetetraploid plants taking place. Fruits were harvested in different daysconsidering the relative maturity to allow full development of seedparts. Seeds were extracted and the number of fruits, total seed weight,average seed weight per fruit, total seed numbers, and average seednumber per fruit determined. The results are presented in Table 3.

TABLE 3 Increased seed weight and seed numbers per fruit resulting fromgrafting of seed parents Total Avg. seed Seed Wt. Per Total Average SeedExperimental Number Weight fruit Seed Number per Material of Fruits(Gms) (Gms) Count fruit B64NG 20 172.86 8.64 2,432 121 B64G 19 182.599.61 2,825 148 1440NG 27 324.17 12 4,078 151 1440G 20 267.63 13.38 3,677184 1009NG 19 176.82 9.3 2,297 121 1009G 15 121.02 8.07 1,854 124 1018NG24 191.32 7.97 2,521 105 1018G 15 130.67 8.71 1,807 120 NG—Non-grafted;G—Grafted

As shown in Table 3, grafted seed parents yielded greater weight andseed number per fruit relative to non-grafted seed parents. For example,the grafted B64 seed parent yielded 9.61 gms of seed per fruit, but thenon-grafted B64 yielded only 8.64 gms of seed per fruit. Likewise, thegrafted 1440 and 1018 plants yielded more seed weight per fruit relativeto their non-grafted plants. The seed weight for grafted 1009 plants waslower than non-grafted plants, possibly because the trait controllingseed number per plant was segregating in this line and was unevenlyrepresented between those plants selected for grafting relative tonongrafting. In addition, the grafted B64 seed parent yielded 148 seedsper fruit, while the non-grafted B64 yielded only 121 seeds per fruit,and each of the grafted 1440, 1009, 1018 plants yielded more seeds perfruit relative to their non-grafted plants.

Example 4 Seed Production in Melon and Watermelon Using Grafting

An analysis was also carried out on the effect on seed production (ingrams per plant) using grafting with melon and watermelon varieties andvarying rootstocks relative to nongrafted checks. The results of theanalysis are presented below.

Number Seed Trial Hectares Hectares plants that production Grams seed %Identification planned Planned plants planted worked in kilograms perplant germination Melon Grafting Rootstock/Grafted Scion ChilsungShintoza 145-22182 FPO 863991 Graft S6V1 0.34 5440 0.3 4790 92.72 19.394-97 Non-graft check S7V5, 6 0.3 4790 77.82 16.25 94-98Rootstock/Grafted Scion Gourd/145-23190 FPO 895010 Graft S5V1 0.46 73600.06 1000 3.68 3.68 87-96 Non-graft check L2V4 0.28 4602 42.07 9.14 95-100 Rootstock/Grafted Scion Chilsung Shintoza 145-23269 FPO 884417Graft S6V1 0.59 9440 0.38 6043 7.24 1.2 42-84 Non-graft check S3V5, 60.38 6043 73.61 12.18 71-94 Rootstock/Grafted Scion Shintoza 145-23206FPO 897683 Graft L6V3 0.33 5280 0.1 1628 22.03 13.53 1628 Non-graftcheck S9V4, 5 0.1 1628 18.66 11.46 1628 Rootstock/Grafted Scion Shintoza145-23304 FPO 880342 Graft S5V1 0.16 2560 0.13 2136 31.1 14.56 73-93Non-graft check L5V3, 4 0.13 2136 36.5 17.08 96-99 Rootstock/GraftedScion Shintoza 145-23249 FPO 872648 Graft S5V1 L6V3 0.33 5280 0.11 182918.26 9.98 84-86 Non-graft check L2V2 0.11 1829 28.85 15.77 84-98Rootstock/Grafted Scion Shintoza 145-22189 FPO 909292 Etapa 14 Graft L6V 4 0.59 9440 0.25 4053 67.95 16.76 90-93 Non-graft check LB, C V 0.251750 16.7 9.5 82-94 18; 18, 19 Rootstock/Grafted Scion Shintoza145-22189 FPO 909292 Etapa 15 Graft DV19; S11V4, 5 0.2 3200 0.11 169720.3 11.96 84-95 Non-graft check CV20, 21, 22 0.11 1697 20.4 12.02 63-90Gal Graft L.6V4 0.19 Rootstock/Grafted Scion Shintoza 145-23377 FPO897686 Graft L6V3, 4 0.44 7040 0.18 2873 25.68 8.94 88-91 Non-graftcheck S 10V4, 5 0.18 2873 18.09 6.3 48-95 Rootstock/Grafted ScionShintoza 145-23269 FPO 897686 Graft S2V2, 3; DV19 0.14 2240 0.09 141115.7 11.12 88-94 Gal Non-graft check CV27 Gal 0.09 1411 22.53 15.9668-94 Rootstock/Grafted Scion Shintoza 145-311134 FPO 909289 Graft DV19Gal 0.1 1600 0.08 1267 15.25 12 93-95 Non-graft check CV27 Gal 0.08 126724.51 19.34 94-95 Rootstock/Grafted Scion Shintoza 145-297888 FPO 917028Graft S2V2, 3; DV18 0.36 5760 0.27 4103 49.29 12 96-95 Gal Non-graftcheck Galmo DV18 0.27 4103 60.78 14.81 85-93 Rootstock/Grafted ScionRootstock/145-23269 FPO 925514 Charmtoza S11V4, 5 0.07 288 0.007 1110.67 6 Chilsung Shimtoza S11V4, 5 480 0.012 205 0.54 2.63 ShimtozaS11V4, 5 460 0.01 169 0.09 0.53 Non-graft check S11V4, 5 34023 494.3214.53 Grafted Scion Rootstock/145-23269 FPO 925514 Hwangtoza S10V2, 30.59 0.05 726 5.32 7.32 Shimtoza (0) S10V2, 3 0.02 311 1.77 5.69Shimtoza (1) S10V2, 3 0.008 138 1.55 11.23 145-22182 (IMPAC) S10V2, 30.11 1717 23.2 13.5 Non-graft check S10V2, 3 34023 494.32 14.53Watermelon Grafting Rootstock/Grafted Scion Chilsung Shintoza/177-21153FPO 880345 Graft S4V1 0.58 9280 0.33 5284 201.83 38 98-99 Non-graftcheck L9V14 Gal 0.33 5284 50.4 9.5 95-98 Rootstock/Grafted ScionChilsung Shintoza/177-25423 FPO 892024 Graft S4, 3V1; 1 0.24 3840 0.152367 41.29 17.4  99-100 Non-graft check L12V4 Gal 0.15 2367 19.26 8.1 99-100 Rootstock/Grafted Scion Shintoza/177-152185 FPO 897682 GraftS3V1 0.08 1280 0.05 863 3.45 3.99 96-99 Non-graft check L9V8, 9 Gal 0.05863 6.12 7.09 97-99 Rootstock/Grafted Scion Shintoza/177-268382 FPO926134 Graft L8V7 Gal 0.1 1600 0.05 810 8.63 10.65 99-99 Non-graft checkL8V7, 8 Gal 0.05 810 4.07 5.02 99-99 Rootstock/Grafted Scion Shintoza177-261691 FPO 909287 Graft L8V9 Gal 0.1 1600 0.06 981 8.53 8.69 98-99Non-graft check L8V9, 10 Gal 0.5 8000 0.06 981 1.16 1.18 99-99Rootstock/Grafted Scion Shintoza 178-295191 FPO 909287 Graft L8V5 Gal.0.01 166 0.01 166 0.29 1.75 Non-graft check L8V5, 6 Gal. 0.01 166 0.291.75 Rootstock/Grafted Scion Shintoza 178-213828 FPO 926141 Graft L8V2,3 Gal. 0.17 2720 0.06 998 0.41 0.41 Non-graft check L8V4, 5 Gal. 0.06998 0.38 0.38

Example 5 Further Analysis of the Effect of Grafting on Seed Production

Grafted diploid watermelon varieties Starbrite and Susanita were graftedon rootstock varieties Charmtoza, Chilsung Shintoza, FR-Couple, andHwangtoza. Seed yield per plant was measured in grams and indicated anincrease in the seed yield per plant for most grafted combinations incomparison with the ungrafted check.

FRUIT YIELD/ VARIETY ROOTSTOCK COUNT YIELD PLANT STARBRITE CHARMTOZA 7451 64.43 CHECK 57 2145 37.63 CHILSUNG SHINTOZA 28 1643 58.68 FR-COUPLE56 2118 37.82 HWANGTOZA 47 2407 51.21 SUSANITA CHARMTOZA 60 1314 21.90CHECK 69 1170 16.96 CHILSUNG SHINTOZA 67 1584 23.64 FR-COUPLE 64 137621.50 HWANGTOZA 55 1409 25.62

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 4,936,904; U.S. Pat. No. 6,018,101-   Cushman, Univ. of Florida, Extension Publication, HS1075, 1-5, 2006.-   Eigsti, HortScience, 6:1-2, 1971.-   Kihara, Proceedings of American Society for Horticultural Science,    58:217-230, 1951.-   Kurata, HortScience 29(4):235-239, 1994.-   Love et al., Euphytica, 35:633-638, 1986.-   Zhang et al., Cucurbit Genetics Coop., 17:111-115, 1994a.-   Zheng et al., J. Heredity, 85(4):279-285, 1994b.-   Zheng et al., HortScience, 31(1):29-57, 123-126, 1996.

1. A method of producing triploid watermelon seed comprising: (a)obtaining a tetraploid watermelon plant, wherein the plant has beengrafted onto a rootstock that exhibits stress tolerance when understress conditions; (b) allowing the tetraploid watermelon plant to bepollinated by a diploid watermelon plant; and (c) allowing triploid seedto form on the tetraploid watermelon plant.
 2. The method of claim 1,wherein the stress tolerance comprises an abiotic stress toleranceselected from the group consisting of cold tolerance, high temperaturetolerance, drought tolerance, and salt tolerance.
 3. The method of claim1, wherein the stress tolerance comprises a biotic stress toleranceselected from the group consisting of a disease resistance, an insectresistance, and a nematode resistance.
 4. The method of claim 1, whereinthe rootstock is from a plant selected from the group consisting ofwatermelon, squash, pumpkin, wax gourd, and bottle gourd.
 5. The methodof claim 1, wherein the diploid watermelon plant is grafted onto arootstock that exhibits stress tolerance when under stress conditions.6. The method of claim 1, wherein the diploid watermelon plant has notbeen grafted.
 7. The method of claim 1, further comprising the step of:(d) selecting the triploid seed from a population of seed resulting fromstep (c) based on the thickness and/or weight of the triploid seed. 8.The method of claim 1, wherein the tetraploid watermelon plant is grownin the absence of said stress conditions.
 9. The method of claim 1,wherein the tetraploid watermelon plant is grown in the presence of saidstress conditions.
 10. The method of claim, wherein the stress tolerancecomprises a biotic stress tolerance selected from the group consistingof viral disease resistance, bacterial disease resistance, and fungaldisease resistance.
 11. The method of claim 7, wherein the step ofselecting is performed manually.
 12. The method of claim 7, wherein thestep of selecting is automated.
 13. The method of claim 1, wherein thetetraploid watermelon plant is pollinated by an insect.
 14. The methodof claim 1, wherein the tetraploid watermelon plant is hand-pollinated.15. The method of claim 1, wherein the grafting is performed by a methodselected from the group consisting of splice grafting, bud grafting,cleft grafting, side grafting, approach grafting, and hole insertiongrafting.
 16. The method of claim 1, wherein the triploid watermelonseed formed in step (c) exhibits a morphology that is distinguishablefrom tetraploid seed produced by the seed parent.
 17. The method ofclaim 1, wherein the tetraploid watermelon plant produces a greaternumber of seed per fruit and/or with a higher degree of hybridityrelative to a watermelon plant of the same genotype as said tetraploidwatermelon plant that has not been grafted and is grown under the sameconditions as the tetraploid watermelon plant.
 18. The method of claim1, wherein the tetraploid watermelon plant is exposed to at least afirst biotic and/or abiotic stress prior to triploid seed forming.
 19. Amethod of producing diploid watermelon seed comprising: (a) obtaining afirst diploid watermelon plant, wherein the plant has been grafted ontoa rootstock that exhibits stress tolerance when under stress conditions;(b) allowing the first diploid watermelon plant to be pollinated by asecond diploid watermelon plant; and (c) allowing diploid watermelonseed to form on the first diploid watermelon plant.
 20. The method ofclaim 19, wherein the stress tolerance comprises an abiotic stresstolerance selected from the group consisting of cold tolerance, hightemperature tolerance, drought tolerance, and salt tolerance.
 21. Themethod of claim 19, wherein the stress tolerance comprises a bioticstress tolerance selected from the group consisting of a diseaseresistance, an insect resistance, and a nematode resistance.
 22. Themethod of claim 19, wherein the first diploid watermelon plant and thesecond diploid watermelon plant are of a different genotype.
 23. Themethod of claim 19, wherein the rootstock is from a plant selected fromthe group consisting of watermelon, squash, pumpkin, wax gourd, andbottle gourd.
 24. The method of claim 19, wherein the first diploidwatermelon plant is grown in the absence of said stress conditions. 25.The method of claim 19, wherein the first diploid watermelon plant isgrown in the presence of said stress conditions.
 26. The method of claim19, wherein the first diploid watermelon plant produces a greater numberof seed per fruit and/or higher degree of hybridity relative to awatermelon plant of the same genotype as said first diploid watermelonplant that has not been grafted and is grown under the same conditionsas the first diploid watermelon plant.
 27. The method of claim 19,wherein the second diploid watermelon plant is grafted onto a rootstockthat exhibits stress tolerance when under stress conditions.
 28. Themethod of claim 19, wherein the second diploid watermelon plant is notgrafted.
 29. The method of claim 19, wherein the first tetraploidwatermelon plant is pollinated by an insect.
 30. The method of claim 19,wherein the first tetraploid watermelon is hand-pollinated.
 31. Themethod of claim 19, wherein the grafting is performed by a methodselected from the group consisting of splice grafting, bud grafting,cleft grafting, side grafting, approach grafting, and hole insertiongrafting.
 32. The method of claim 19, wherein the first diploidwatermelon plant is exposed to at least a first biotic and/or abioticstress prior to seed forming.
 33. A method of producing plant seedcomprising: (a) obtaining a first plant, wherein the plant has beengrafted onto a rootstock that exhibits stress tolerance when understress conditions; (b) allowing the first plant to be pollinated by asecond plant of the same species; and (c) allowing seed to form on thefirst plant, wherein the first plant is grown in the absence of saidstress conditions.
 34. The method of claim 28, wherein the first andsecond plants are of a species selected from the group consisting of hotpepper, sweet pepper, tomato, squash, cucumber, pumpkin, gourd, melon,eggplant, and okra.
 35. The method of claim 28 wherein the stressconditions comprise bacterial, fungal and/or viral disease.
 36. Themethod of claim 28, wherein the first plant produces a greater number ofseed per fruit and/or higher degree of hybridity relative to a plant ofthe same genotype as said first plant that has not been grafted and isgrown under the same conditions as the first plant.
 37. A population ofplants planted in pollinating proximity in a field comprising: (a)tetraploid watermelon seed parent plants, wherein the plants have beengrafted onto a rootstock that exhibits stress tolerance when understress conditions; and (b) diploid watermelon pollen donor plants. 38.The population of claim 37, wherein the tetraploid watermelon seedparent plants and diploid watermelon pollen donor plants are planted inalternating rows.
 39. The population of claim 38, wherein the rows areplanted in a ratio of rows comprising tetraploid watermelon seed parentplants to diploid watermelon pollen donor plants of 1:1, 2:1, 3:1, 4:1or 5:1.